Tad pili contribute to the virulence and biofilm formation of virulent Aeromonas hydrophila

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170 A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.


Introduction
Aquaculture helps meet the ever-growing global demand for animal protein, yet it faces a substantial threat from fish pathogens, leading to economic losses and food insecurity (Mzula et al., 2019;Irshath et al., 2023).Among these pathogens, several Aeromonas species are prominent agents of Motile Aeromonas Septicemia (MAS) in temperate and warm freshwater fish, with Aeromonas hydrophila being a significant contributor (Irshath et al., 2023).
A third subclass of T4P, Type IV class c pili (T4cP), was recently classified due to the distinct genetic organization and evolutionary history of Tight Adherence Pili (Tad) (Beeby, 2019;Denise et al., 2019).Tad pili, originally discovered in Actinobacillus actinomycetemcomitans, are crucial for forming bundled fibers that facilitate autoaggregation, adherence, colonization, and biofilm development (Kachlany et al., 2000).It has been postulated that Tad originated via horizontal acquisition from Archaea given their significant similarity to archaeal Epd-like systems (Denise et al., 2019).For example, Tad, located on single locus, lacks a retraction ATPase and instead encodes a bifunctional ATPase responsible for both extension and retraction processes (Beeby, 2019;Ellison et al., 2019;Piepenbrink, 2019).
A. hydrophila exhibits outer membrane-spanning type I pili and T4P. A. hydrophila encodes two distinct types of T4aP: Mannose-Sensitive Hemagglutinin (MSHA) and Type IV Aeromonas Pilus (Tap).MSHA bundle forming pili shares high homology to the MSHA pili of Vibrio spp., while Tap pili shares high homology to the Pil system of Vibrio and Pseudomonas spp.In addition, previous research reported the consistent presence of genes encoding Tad pili in vAh genomes (Tekedar et al., 2019).Notably, the Tad cluster was also found in a clinical A. hydrophila isolate infecting humans, suggesting potential significance in pathogenicity (Tekedar et al., 2019).However, within the context of vAh pathogenicity, the precise functions of the T4P have not been investigated previously.
The Tad system, with its diverse functions, serves as a bridge that facilitates interaction between the pathogen and its host during infection.This study aimed to delineate the function of Tad pili in vAh isolate ML09-119.We conducted an expanded comparative genomics analysis involving 170 A. hydrophila genomes sourced from the National Center for Biotechnology Information (NCBI), confirming the consistent presence of the Tad gene cluster in vAh genomes.We constructed a mutant strain with complete deletion of the Tad operon and investigated its contribution to virulence in catfish.Additionally, the influence of Tad pili on A. hydrophila physiology and response to environmental stress was determined.These results revealed the significant role of Tad pili in the virulence of vAh isolate ML09-119, highlighting its crucial function in mediating pathogenic interactions.

Genome features and building phylogenetic tree
Genome sequence data for 170 A. hydrophila genomes were downloaded from the NCBI database.Genome metadata is listed in Table 1.All selected genomes shared at least 95% average nucleotide identity (ANI) among them, indicating conspecificity, and a phylogenetic tree was built from the core genome (Blom et al., 2016).Also, AAI was calculated with EDGAR 2.0 (Blom et al., 2016).Gene sets from the core genome were aligned individually using MUSCLE (Edgar, 2004) then concatenated.The concatenated alignment was used to compute a Kimura distance matrix, which was served as input for the Neighbor-Joining algorithm implemented in PHYLP (Baum, 1989).The phylogenetic tree for 170 genomes was build out of a core of 1893 genes per genome, 321810 in total.The core has 1711702 AA-residues/bp per genome, 290989340 in total.

Distribution of individual Tad operon genes in 170 A. hydrophila genomes
The virulence factor database (VFDB) (Liu et al., 2022) and MacSyFinder tool were used with default features to identify individual Tad operon genes across the 170 A. hydrophila genomes investigated (Abby et al., 2014;Abby et al., 2024).Specifically, the "unordered" dataset option was chosen because most of the evaluated genomes were incomplete drafts.Briefly, the following parameters were used: the topology of the replicon was linear/circular, maximal E-value was 1.0, maximal independent Evalue was 0.001, and the minimal profile coverage was 0.5.Both mandatory genes and accessory genes were identified.The protein   sequences of the 13 Tad operon genes were identified using VFDB and MacSyfinder.Based on these findings, the 13 genes were organized in an operon structure (Figure 1).The 13 Tad operon gene protein sequences were then uploaded to CLC Genomics Workbench (version 23.0.3),where BLASTp searches were conducted to compare the 13 Tad genes against the 170 A. hydrophila genomes.Only matches with E-values 1×10 -20 were considered present.The distribution of the predicted 13 Tad operon genes among 170 A. hydrophila genomes was presented using the pheatmap library in R Studio (Version 2023.06.0 + 421) (Figure 2).Also, the GC content of the Tad operon was analyzed and compared to the entire genome of A. hydrophila ML09-119 using SnapGene (version 6.2.1) (Figure 1).

Bacterial strains and plasmids
A. hydrophila strain ML09-119 (Tekedar et al., 2013) was used as a representative of the vAh clonal group impacting U.S. channel catfish aquaculture.The bacterial strains and plasmids used in this study are listed in Table 2. Escherichia coli strains CC118 lpir and BW19851 were used for cloning and conjugation purposes, respectively, and suicide plasmid pMEG-375 was used to transfer gene mutations into A. hydrophila.Brain heart infusion (BHI) agar and broth (Difco, Sparks, MD, USA) were used to grow A. hydrophila strain at 30°C.Luria-Bertani (LB) agar and broth (Difco) were used for E. coli incubation at 37°C.When needed, the following antibiotics and reagents (Sigma-Aldrich, Saint Louis, MN, USA) were used in the given concentrations: colistin (12.5 mg/ mL), chloramphenicol (10-25 mg/mL), ampicillin (100 µg/mL), mannitol (0.35%), and sucrose (5%).

In-frame deletion of Tad operon in A. hydrophila ML09-119
In-frame deletion was used to create a complete Tad operon mutant in A. hydrophila ML09-119.Briefly, the Tad operon mutant was constructed by allelic exchange and homologous recombination using suicide plasmid pMEG-375 containing the counter selectable gene sacB (Dozois et al., 2003).Four primers (A, B, C, and D) were designed for the Tad operon region using Primer3 (https://primer3.ut.ee/) (Untergasser et al., 2012) (Table 3).Two specific compatible restriction enzyme sites were introduced in A and D primers for cloning, and the reverse complement of primer B was added to the 5′ end of primer C to create an overlap region to initiate the fusion of PCR fragments by overlap extension PCR (Horton et al., 1989).The upstream (AB fragment) and downstream (CD fragment) regions of the Tad operon were amplified using two sets of primers.Amplified PCR fragments AB and CD were annealed at the overlapping regions, and a single fragment was created using the A and D primers.The AD product was purified, digested, and ligated into digested pMEG-375, then transformed into E. coli CC118lpir, and the final product was grown on LB agar with ampicillin.The positive plasmids carrying the mutated Tad operon were transferred into A. hydrophila ML09-119 by conjugation using E. coli BW19851 strain.Transconjugants were selected on plates containing chloramphenicol and colistin.In this process, chloramphenicol was used to identify the integration of pMEG-375 in A. hydrophila ML09-119 chromosome, while colistin was used as counterselection against E. coli.For confirmation purposes, PCR was used to verify the vector had integrated correctly into the A. hydrophila ML09-119 chromosome.Following sucrose selection, colistin resistant and chloramphenicol sensitive colonies were selected, and the candidate mutants were screened by colony PCR using A and D primers.Mutant validation was done by sequencing AD fragments amplified from chloramphenicol sensitive mutants using Tad-ConF01 and Tad-ConR01 (Table 3).The final A. hydrophila Tad operon mutant was designated as vAhDtad.

Verification of Tad mutant, vAhDtad, by complete genome sequencing
The vAhDtad was expanded in 9 mL porcine brain heart infusion broth (Becton Dickinson, Franklin Lakes, NJ, USA) in static, overnight cultures at 28°C.An aliquot (3 mL) of the expanded culture was pelleted by centrifugation (20,000 x g).High molecular weight genomic DNA was isolated from the concentrated pellet using the PureGene DNA isolation kit (QIAGEN, Hilden, Germany).Long reads were produced on a GridION platform (Oxford Nanopore Technologies, Oxford, UK) using the rapid barcoding kit (RBK004) and v9.4.1 flow cells.These sequences were filtered to an average quality 10 using NanoFilt v2.2.0 (Bolger et al., 2014) to produce 211,519,148 bases from 30,912 reads of at least 1000 bp with an average read length of 6842 bp.The genome was assembled into a single contig with Canu v1.7 (Koren et al., 2017), the consensus sequence was produced using Medaka v0.11 (Oxford Nanopore Technologies, https:// github.com/nanoporetech/medaka),and the contig was circularized manually.

Virulence of the vAhDtad mutant in catfish fingerlings
All fish experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Mississippi State University.Virulence of vAhDtad was compared to A. hydrophila ML09-119 wild-type strain (vAh-WT) by immersion route of exposure as described previously (Abdelhamed et al., 2016).Briefly, specific-pathogen-free (SPF) channel catfish fingerlings (13.54 ± 0.54 cm, 15.32 ± 1.98 g) were stocked into twelve 40-liter flow-through tanks (14 fish/tank) and acclimated for a week.Tanks were assigned randomly to three treatment groups: vAhDtad, vAh-WT, and BHI (sham infected).Each group included three replicate tanks.Fish were fed twice daily with a commercial catfish feed, and water temperature was maintained at 30°C (±2) throughout the experiments.On the challenge day, the water levels in each tank were decreased to 5 L, and 100 mL of overnight culture were added directly to each tank (1.9 × 10 9 CFU/mL water).After three hours, the water flow was restored, and fish were maintained as usual.Negative control (sham-infected group) tanks were treated similarly, but exposed to 100 mL of sterile BHI broth.During the immersion process, the water was well aerated.Fish mortalities were recorded daily for 7 days, and cumulative percent mortality calculated for each group.

Growth kinetics
Growth kinetics of vAhDtad and vAh-WT in BHI broth were compared.Briefly, bacterial growth curves were generated by measuring the optical density at 600nm (OD 600 ) on a multimode reader (BioTek Cytation 5) every half an hour for 72 h at 30°C.The growth assays were conducted in two independent experiments, each with 24 replicates for vAhDtad and vAh-WT strains.

Biofilm formation
Biofilm formation of the vAhDtad and vAh-WT strains were compared using a microtiter plate assay.Five different colonies of each strain were cultured to log-phase (OD 600 = 0.6 ± 0.05) at 28°C in BHI broth and diluted to OD 600 of 0.1 using freshly prepared media.A 100 µL of the diluted cultures were added into a 96-well plate and incubated at 28°C overnight.Sterile BHI media (100 µL) was added to the blank control wells.For quantitative analysis, 8 replicate wells were used for each treatment and the control group.After incubation, the bacteria were discarded by turning the plate over and shaking out the liquid, then gently submerging the plate in a tub of water.The washing step was repeated twice to remove any unbound bacteria and media before staining.To each well, 125 µL of crystal violet (CV) (0.1%) was added and the plate was incubated at room temperature for 15 min.Next, the microtiter plate was rinsed 3-4 times by submerging in sterile water as described above, followed by vigorous blotting on a stack of paper to remove any unbound bacteria and dye.The plate was air-dried and 125 µL of 95% ethanol was added to each well to solubilize the CV.The plate was incubated for 15 min and the solubilized CV was transferred to a new flat bottom microtiter plate and absorbance was measured at 595nm.The biofilm assay was conducted with 35 biological replicates for both vAhDtad and vAh-WT strains.

Motility
The vAhDtad and vAh-WT strains were cultured to log-phase (OD 600 = 0.6 ± 0.05) at 28°C in BHI broth.Two microliters of bacterial culture were spotted on a 0.6% BHI semi-solid plate and incubated at 28°C.Replicate plates were used for statistical analysis.The diameters of bacterial growth were measured after 16 hours of incubation.Each motility assay was conducted with 7 independent biological replicates, with 3 technical replicates per biological replicate, for both the vAhDtad and vAh-WT strains.

Temperature stress
The ability of the vAhDtad and vAh-WT strains to withstand extreme temperatures was evaluated.Both strains were cultured to log phase (OD 600 = 0.6 ± 0.05) at 28°C and then adjusted to an OD 600 of 0.1 using sterile BHI broth.The bacterial suspension was subsequently incubated at 18°C, 28°C (control), and 37°C for 24h at 180 rpm.Following incubation, the cultures were washed twice with sterile phosphate-buffered saline (PBS) and spread on BHI agar plates for colony counting and statistical analysis.The temperature assay was conducted with 4 biological replicates for both vAhDtad and vAh-WT strains.

Acid-alkali stress
The vAhDtad and vAh-WT strains were cultured to log-phase (OD 600 = 0.6 ± 0.05) at 28°C in BHI broth.Bacterial pellets were collected, washed twice with sterile PBS, and incubated in BHI at pH 5.0, pH 6.0, pH 7.0 (control), pH 9.0, and pH 10.0 for 30 minutes at 28°C.Viable bacteria were quantified by spreading on BHI agar plates after serially diluting.The two strains' percent viability (CFUs/mL) was calculated and compared.The acid-alkali stress assay was conducted with 3 biological replicates for both vAhDtad and vAh-WT strains.

Osmotic stress
Under optimal growth conditions at 28°C, the vAhDtad and vAh-WT strains were cultured to log-phase with an optical density of 0.6 ± 0.05 at 600nm.Bacterial pellets were collected, washed with PBS, and resuspended in fresh BHI broth containing 0.5 M sodium chloride (NaCl), which was then adjusted to OD 600 of 0.1.Subsequently, 200 mL of each bacterial suspension was dispensed into separate wells of a 96-well microplate.To the control wells, 200 mL of sterile BHI broth was added.Growth was monitored every 1 h for 16 h at 28°C in an automatic microplate reader using absorbance at 600nm.The osmotic stress assay was conducted with four biological replicates for both vAhDtad and vAh-WT strains.

Oxidative stress
To evaluate the viability of the strains against environmental oxidants, both vAhDtad and vAh-WT strains were cultured to logphase (OD 600 = 0.6 ± 0.05) at 28°C in BHI broth.Bacterial pellets were collected and washed twice with sterile PBS solution before treatment with 0.1M hydrogen peroxide (H 2 O 2 ), using PBS as control.Bacteria were incubated for 20 min at 28°C, and the viable bacteria were quantified by spreading them onto BHI agar.The percentage viability of the two strains in the treatment group (H 2 O 2 ) compared to the control group (PBS) was calculated and compared between the mutant and wild-type strain.The oxidative stress assay was conducted with four biological replicates for both vAhDtad and vAh-WT strains.

Scanning electron microscopy
The effects of Tad pili on the membrane structure of A. hydrophila was evaluated using scanning electron microscopy.Both the vAhDtad and vAh-WT strains were grown at 30°C.Pellets were collected from log phase and overnight cultures, and bacteria were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate buffer for 2 hours at room temperature prior to fixation in 1% osmium tetroxide for one hour.Fixed pellets were then dehydrated in a graded ethanol series (10%, 20%, 30%, 50%, 70%, 80%, 85%, 95%, 100%), dried via a critical point dryer, mounted on aluminum stubs, and sputter coated with 30nm platinum.Images were captured using a JEOL 6500F field emission SEM operating at 10KeV at the Mississippi State University Institute for Imaging and Analytical Technologies.

Statistical analysis
Mean cumulative percent mortality data were arcsine transformed, and analysis of variance (ANOVA) was applied using PROC GLM in SAS for Windows v9.4 (SAS Institute, Inc., Cary, NC).For the characterization assays, the colony forming units/mL between the vAhDtad and vAh-WT strains were compared and analyzed using one-way analysis of variance (ANOVA).An alpha level of 0.05 was used in all analyses.

Distribution of Tad operon in A. hydrophila isolates
This study comprehensively analyzed 170 A. hydrophila genome sequences derived from diverse geographical locations and hosts (Table 1), several of which originated from catfish disease cases in Alabama and Mississippi (Tekedar et al., 2013;Tekedar et al., 2015;Tekedar et al., 2016a;Tekedar et al., 2016b;Tekedar et al., 2017).Isolate identity was confirmed by ANI, and all selected genomes displayed ANI values of 95% or higher, indicating they are all the same species (Goris et al., 2007).Subsequently, a phylogenetic tree was constructed for the 170 A. hydrophila strains based on a core set of 1893 genes per genome, totaling 321,810 genes.The core genome contained 1,711,702 amino acid residues per base pair or 290,989,340 in total.Notably, the vAh isolates formed a distinct cluster with 100% branch conservation, and this separation was verified by ANI and average amino acid identity (AAI) values (data not shown here).

Tad operon deletion impairs virulence and biofilm formation of A. hydrophila ML09-119
To investigate the role of Tad pili in A. hydrophila ML09-119 virulence, infectivity trials were conducted in catfish fingerlings.The vAhDtad strain exhibited a significantly lower mortality rate (14.65%) compared to the vAh-WT strain (74.36%) 72 hour postinfection (Figure 3A).Next, the impact of deletion of the Tad operon on growth kinetics of vAh isolate ML09-119 in BHI broth was assessed, revealing no measurable differences in growth (Figure 3B).Furthermore, the role of the Tad pili on biofilm formation was evaluated.The vAhDtad strain displayed significantly reduced (p ≤ 0.05) biofilm formation compared to the vAh-WT strain (Figure 3C), indicating that Tad pili are crucial for biofilm formation.No significant difference in motility was detected between the mutant and wild type parent (Figure 3D).

Role of Tad pili in stress resistance
The influence of Tad in the tolerance of A. hydrophila to environmental stressors, including temperature, pH, NaCl, and H 2 O 2 was evaluated.The vAhDtad mutant strain exhibited growth to a higher density at 28°C than vAh-WT strain, but there were no observed differences in growth at 18°C and 37°C (Figure 4A).Under acid-alkali stress, both strains demonstrated similar growth at neutral pH 7.0, with a baseline percent viability of 100% (Figure 4B).However, as the pH deviated from neutrality, distinct trends emerged.Under acidic conditions (pH 5.0) the vAhDtad strain demonstrated significantly reduced viability compared to the vAh-WT strain (p ≤ 0.05) (Figure 4B).Under osmotic stress conditions, no significant difference was observed in the growth kinetics between the vAhDtad mutant and the vAh-WT strain.However, the vAhDtad strain had a shorter log-phase than the vAh-WT strain, suggesting a potential adaptation that renders it slightly quicker to adapt to osmotic stress (Figure 4C).Following exposure to hydrogen peroxide (H 2 O 2 ), both the vAhDtad mutant and the vAh-WT strain exhibited decreased viability, with no observed differences between the mutant and the wild-type, indicating that Tad does not confer tolerance to oxidative stress in A. hydrophila (Figure 4D).Collectively, these results indicate an impact of Tad pili on acid tolerance in vAh strain ML09-119, but not temperature or osmotic stress.

Tad pili affect bacterial surface structure in A. hydrophila ML09-119
Scanning electron microscopy was used to evaluate the effects of Tad pili on the surface structure of A. hydrophila ML09-119.While overnight cultures showed no apparent differences (data not Evaluating the role of Tad operon in A. hydrophila ML09-119.The vAhDtad and vAh-WT strains were compared and analyzed using one-way analysis of variance (ANOVA).An alpha level of 0.05 was used for all analyses, with asterisks (*) indicating statistical significance.(A) Percent mortalities of fish immersion challenge.The fish were exposed to 2 treatments, A. hydrophila and vAhDtad.Fish mortalities were recorded daily for 7 days, and percent mortalities was calculated for each treatment.(B) Growth Curve.Bacterial curves were generated by measuring the optical density at 600nm (OD 600 ) on a multimode reader every half an hour for 72h at 30°C.(C) Biofilm assay.The strains were cultured to log phase and adjusted to OD 600 of 0.1.The cultures were added to 96-well plate and incubated at 28°C for 24h.The cultures were washed and air-dried post staining with crystal violet (CV) for 15min at room temperature.The CV was solubilized with 95% ethanol for 15min, and absorbance was measured at 595nm.(D) Motility assay.The strains were cultured to log-phase.Two microliters of the culture were spotted on 0.6% BHI semi-solid plates and incubated at 28°C for 24h.The motility diameter for both strains were measured in millimeters.Tekedar et al. 10.3389/fcimb.2024.1425624Frontiers in Cellular and Infection Microbiology frontiersin.orgshown), we observed changes in the bacterial surface morphology during the log-phase.In the log phase, vAh-WT strain displayed a distinctly smooth material covering its surface, a feature absent in the vAhDtad, which exhibited a surface that appeared rough (Figures 5B, D).In addition, the vAh-WT strain possessed prominent protrusions on its surface, while vAhDtad lacked these distinctive surface features (Figures 5A, C).

Discussion
Amidst the dynamics of host-pathogen interactions, pathogens are driven to persistently evolve, adapt to varied ecological niches, and evade the host's immune defenses.These adaptations often occur by acquiring unique genes, toxins, and systems through horizontal gene transfer, gene fissions, or gene duplications, which leaves a distinct imprint on the pathogen's genetic makeup.Comparative genomics allows identification of these unique genetic adaptations, which can be specific for a bacterial species or for a genetic lineage within a species.A. hydrophila is a genetically diverse species that has many unique lineages with varied environmental adaptations, virulence, and host specificities.
Based on previous work, vAh isolates consistently encode a unique T4cP, known as tight adherence (Tad), which is largely absent from non-vAh isolates (Tekedar et al., 2019).Based on this key finding, it was hypothesized that acquisition of the Tad operon is an adaptive advantage, potentially contributing to the pathogenicity of vAh isolates.To test this hypothesis, a comprehensive comparative genome analysis was conducted with 170 A. hydrophila genomes, sourced from diverse hosts and distinct geographical locations.All evaluated genomes shared an ANI ≥95%, indicating conspecificity (Kim et al., 2014).A core-genome based phylogenetic tree revealed that vAh isolates from different geographical origins form a discrete subgroup (Hossain et al., 2014;Tekedar et al., 2019).These analyses also support the notion that U.S. and Chinese vAh isolates originated from the same monophyletic clade.Furthermore, in silico analysis confirmed previous findings that vAh isolates consistently carry the Tad operon.
Homologs of Tad loci are widely distributed across plant, human, and animal pathogens such as Sinorhizobium meliloti (Zatakia et al., 2014), Yersinia pestis (Tomich et al., 2007), Bifidobacterium breve (O'Connell Motherway et al., 2011;Ellison et al., 2019;O'Connell Motherway et al., 2019;Alessandri et al., 2021), Vibrio vulnificus (Pu et al., 2018a;Duong-Nu et al., 2019;Zhang et al., 2021), Myxococcus xanthus (Seef et al., 2021), Mycobacterium tuberculosis (Hosseini et al., 2014;Ramsugit and Pillay, 2015;Mann et al., 2016;Alteri et al., 2022), Caulobacter crescentus (Skerker and Shapiro, 2000;Mignolet et al., 2018;Hershey et al., 2019;Sangermani et al., 2019;Mignolet et al., 2021), and Cutibacterium acnes (Davidsson et al., 2017).The presence of the Tad system in such diverse bacterial species hints at an adaptive advantage, substantiated by its diverse roles.Tad pili have been implicated in biofilm formation, which provides a protected environment that enables increased resistance to Characterization of vAhDtad under different stress conditions.For the characterization assays, vAhDtad and vAh-WT strains were compared and analyzed using one-way analysis of variance (ANOVA).An alpha level of 0.05 was used for all analyses, with asterisks (*) indicating statistical significance.(A) Temperature stress assay.The strains were cultured to log phase and OD 600 was adjusted to 0.1 with BHI medium.The bacterial suspension was incubated at 18°C, 28°C (as a control) and 37°C for 24h.The cultures were double diluted with sterile PBS and plated onto BHI agar, for colony counting and statistical analysis.(B) Acid-Alkali tolerance.The strains were cultured at log-phase and the pellet was collected, washed twice with sterile PBS, and subjected to treatment in BHI at pH 4.0, pH 5.0, pH 7.0 (as a control), pH 9.0, and pH 10.0 for 30 minutes at 28°C.Viable cells were counted and compared for both strains by plating on BHI agar after serial dilutions.(C) Osmotic stress assay.The strains were cultured to log phase; pellets were collected and resuspended in BHI with 0.5M NaCl to adjust the OD 600 of 0.1.The OD 600 was monitored every 1h for 16h at 28°C.(D) Oxidative stress assay.The strains were cultured to log phase; pellets were collected and treated for 20min in PBS with 0.1M H 2 O 2 and PBS without H 2 O 2 as a control.Viable cells were counted on BHI plates after incubating for 24h.
In particular, Tad pili serve a unique function in the early stages of infection by acting as an adhesive bridge, facilitating the attachment of pathogens to host cells.For instance, Tad pili are indispensable for Bifidobacterium breve to colonize the human gut.In particular, the tadE gene contributes to host signaling that promotes colonic host epithelial proliferation (O'Connell Motherway et al., 2019).Similarly, in S. meliloti, the Tad locus is necessary for competitive bacterial-plant symbiotic interactions (Zatakia et al., 2014).
In vAh ML09-119, a set of 13 Tad genes organized within an operon structure were identified (Figure 1).The function of Tad in vAh was investigated through the generation of a knockout mutant that encompassed the entire operon.Compared to the wild-type strain, there was significantly reduced mortality in catfish fingerlings exposed to vAhDtad indicating the Tad operon plays a critical role in the virulence of A. hydrophila ML09-119 (Figure 3A).This finding is consistent with the role of the Tad operon in diverse host-pathogen systems.
In contrast to these findings, studies on Aeromonas salmonicida (Boyd et al., 2008) and the human pathogen M. tuberculosis, which primarily focused on disruptions in a subset of the Tad operon genes, revealed the Tad operon was not essential for virulence in those host-pathogen interactions (Mann et al., 2016).Comparably, the current study involved a complete deletion of the Tad operon, resulting in significant attenuation.These contrasting outcomes could be driven by species-or host-specific nuances during hostpathogen interactions.For example, deleting Tad genes in a human pathogen, M. tuberculosis, did not impact biofilm formation or virulence in a mouse model (Davidsson et al., 2017).In contrast, the Tad operon in M. tuberculosis was upregulated upon interaction with human epithelial cells (Alteri et al., 2022), suggesting that differences in host environment may affect the function of Tad pili.Interestingly, Cutibacterium acnes type II isolates carrying plasmids with four Tad genes from human prostate cancer specimens were speculated to trigger infection-induced prostate cancer (Davidsson et al., 2017).Herein, a complete Tad operon was identified from some clinical A. hydrophila isolates from humans in the USA and China (Figure 2), suggesting Tad may facilitate A. hydrophila invasion of human cells.However, despite these observations, the precise role of Tad pili in A. hydrophila isolated from human cases remains a subject for future investigation.
A prominent hypothesis in studies focused on pathogens including A. actinomycetemcomitans, Haemophilus ducreyi, Pasteurella multocida, V. vulnificus, C. crescentus, M. tuberculosis and S. meliloti, suggests the Tad system has been horizontally transferred (Planet et al., 2003;Zatakia et al., 2014;Ramsugit and Pillay, 2015;Pu et al., 2018b;Beeby, 2019;Ellison et al., 2019).Evolutionary analyses suggest this genetic transfer coincided with the development of a 'EppA-dependent' (Epd) pilus homologue, acquiring a secretin, tracing their origins to an ancient genetic exchange with the Archaea (Denise et al., 2019;Sonani et al., 2023).It is thought the Tad operon may provide a competitive edge by facilitating niche adaptation, allowing the pathogen to persist in diverse environments.
Tad systems show considerable diversity among bacterial species, revealing distinct arrangements and functional roles.For instance, M. tuberculosis maintains only five Tad genes, diverging from the typical functional Tad loci which consists of 12-14 genes organized in a single operon.Other organisms like Burkholderia pseudomallei, Mesorhizobium loti, and V. vulnificus harbor three Tad loci (Hossain et al., 2014).In V. vulnificus, the deletion of all three Tad loci was necessary to mitigate pathogenicity (O'Connell Motherway et al., 2011).S. meliloti, on the other hand, encodes two Tad loci, situated in the chromosome and a megaplasmid (Kim et al., 2014).In comparison, C. crescentus has seven genes homologous to the Flp pilus genes; in this species, Flp pili serve as surface sensors altering secondary molecules controlling cell cycle events (Ellison et al., 2017;Sangermani et al., 2019).In Mycobacterium luteus, two Tad clusters located 1.2 Mbp apart are crucial for natural competence (Angelov et al., 2015).
In A. hydrophila ML09-119, the Tad operon has higher GC content (64.82%) than the average for the rest of the genome (60.82%) (Figure 1), suggesting acquisition from horizontal transfer.Interestingly, vAh isolates consistently encode the Tad operon while exhibiting the absence of any type III secretion system (T3SS) (data not shown here).This suggests the mechanism of pathogenesis for vAh, particularly how vAh manipulates function of host cells, is quite different from pathogens that utilize T3SS to inject effector proteins directly into host cells, and Tad may be an important adaptation in the absence of T3SS.
In addition to its role in pathogenicity, the effects of the Tad operon knockout on critical factors such as growth kinetics, motility, and biofilm formation in A. hydrophila was also assessed.Although deletion of the Tad operon had no effect on growth rate and motility, it significantly reduced biofilm formation.Tad pili have been reported as a crucial adhesion factors for initial surface attachment, a vital step in microcolony formation.They facilitate cell-cell interactions and aggregation, promoting the development and maturation of structured biofilms.Biofilms shield pathogens from environmental stressors and host defenses, enhancing survival against antimicrobial peptides and phagocytosis.
The significance of T4P extends beyond their conventional roles in attachment and motility, including the capacity to perceive cues within the environment and orchestrate responses.In the current study, Tad pili did not contribute to A. hydrophila resistance against most stress factors investigated (Figure 4).However, a significant contribution to survival at pH 5 was observed.Interestingly, in the human pathogen M. tuberculosis, acidic pH of the phagosomal vacuole was shown to trigger assembly of the Tad pilin (Alteri, 2005).In A. hydrophila, Tad pili may function as a sensor regulator for low pH, inducing expression of other adaptive mechanisms, or it may directly mediate resistance to low pH.
Structural observations under SEM revealed distinct characteristics in actively dividing A. hydrophila.vAh-WT exhibited a smooth layer absent in vAhDtad bacteria, which displayed rough, irregular protrusions with a coarse texture (Figure 5).Interestingly, these distinct surface modifications were only observed in the log-phase of growth and were absent during the stationary phase.Because Tad pili are a surface structure, it is not surprising that surface alterations were detected by SEM, but the significance of the rough surface during log phase is not currently known.In C. crescentus, Tad pili regulate secondary signaling pathways through mechanical sensing of surface cues, leading to the production of highly efficient progeny equipped for sustained attachment (Sangermani et al., 2019;Snyder et al., 2020).It is possible the bacterial surface alterations in vAhDtad impacted host cell adhesion and/or induction of virulence mechanisms through signaling pathways resulting in reduced virulence, but further research is needed.
Collectively, these investigations into the function of Tad pili in vAh isolate A. hydrophila ML09-119 provides valuable insights into its role in virulence and environmental adaptation.Comparative genomic analysis confirmed the consistent presence of the Tad operon in the vAh subclade of A. hydrophila, suggesting Tad is essential for vAh virulence in catfish, biofilm formation and resistance to acid stress.This work highlights the multifaceted role of Tad pili in A. hydrophila pathogenicity and physiology.The presence of the Tad operon in some human A. hydrophila isolates suggests it may contribute to pathogenesis in humans as well, but further investigation is needed.Regardless, these findings demonstrate a clear role of Tad pili in contributing to virulence of vAh in catfish, which is one of the most important diseases affecting catfish aquaculture in the southeastern United States.Further investigation is warranted to delineate the role of Tad pili in vAh pathogenesis and explore its potential for treatment and prevention of motile Aeromonas septicemia.
FIGURE 1 Genetic map of the A. hydrophila ML09-119 Tad pilus operon and its component genes.(A) GC content analysis of the Tad operon within the A. hydrophila ML09-119 genome.The genome location of the Tad operon is indicated in the figure.(B) The components of the Tad operon, along with their predicted or known functions, are indicated.The Tad genes are color-coded based on their respective functions.

FIGURE 2
FIGURE 2Phylogenetic tree based on the Aeromonas hydrophila core genome and availability of individual Tad operon genes across 170 A. hydrophila genomes.Isolation sources and geographical locations for each genome are included.Navy blue color represents the presence of Tad operon genes, whereas light blue represents absence.Branch lengths were reduced to fit the image.
FIGURE 4 FIGURE 5 Ultrastructural observation of vAhDtad and vAh-WT.Bacterial strains were grown to log-phase in BHI at 30°C and specimens were examined with JEOL 6500F field emission SEM operating at 10 KeV.The white arrows indicate dividing cells.(A) Cell structure of vAhDtad at 18,000x and (B) at 55,000x under SEM.(C) Cell structure of vAh-WT at 18,000x and (D) at 55,000x under SEM.

TABLE 1
Genome features for 170 A. hydrophila genomes utilized in this study.

TABLE 2
Bacterial strains and plasmids used in the present study.

TABLE 3
Primers used to generate and verify in-frame deletion of vAh Tad operon.
Bold letters show restriction enzyme (RE) recognition sequences added to primers.Underlined bold letters indicate reverse complement of Tad-IR primer sequence.